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BDNF Grant Proposal
- 2. A. SPECIFIC AIMS
Major depressive disorder (MDD) is one of the most common and debilitating mental disorders in the world,
affecting approximately 9.1% of the population in any given year (SAMHSA, 2013). One of the strongest known
correlates with depressive symptoms is underexpression of brain-derived neurotrophic factor (BDNF) in the
hippocampus (Boersma, et al. 2013; Grah, et al. 2014). BDNF can be underexpressed if the promoter IV of the bdnf1
gene is highly methylated, a change which has been linked to chronic stress (Gomez-Pinilla, 2011). Understanding
the means by which bdnf methylation changes in response to stress is central to this project. DNA methylation by
DNA methyltransferase 1 (DNMT1) generally reduces transcription and DNA hydroxymethylation (catalyzed by
ten-eleven translocation methylcytosine dioxygenase 1 or TET1) increases transcription (Guo, et al., 2011). If the
balance of DNMTs and TET1 favors methylation for too long, particularly in childhood, there is an increased risk of
developing MDD. This project will examine the effect of acute and chronic stress on the balance of methylated and
non-methylated cytosine (here termed 5mC equilibrium) in the hippocampal neurons of rats. Acute stress refers to
the period in which neural activity related to the stimulus continues, while chronic stress refers to the period
afterward, when only a hormonal stress response continues. We hypothesize that TET1 expression is dependant
on neural activation and DNMT1 expression is dependant on stress hormone signalling, and will approach this
topic with the following aims:
Aim 1: To characterize the expression profile of TET1 in the neonatal rat hippocampus
Our working hypothesis for this aim is that TET1 expression will follow a similar pattern to DNMT expression
reported by Simmons, et al., 2013 in the neonatal rat brain. We predict that in order to maintain a dynamic DNA
methylation pattern in the maturing brain, TET1 will be highly upregulated immediately following birth and will
decrease in expression progressively as maturation progresses. We will measure mRNA expression of tet1
transcripts by qPCR and protein expression by Western blotting. If our prediction is correct, this suggests a
mechanism by which early stress predisposes rats and humans alike to depression later in life.
Aim 2: To examine the effect of acute and chronic stress on BDNF, TET1, and DNMT1 expression
We will address this aim with an in vitro and in vivo model. Our working hypothesis for the in vitro model is based
upon the proposed model in Fig. 1, and predicts that channelrhodopsin-2 expressing hippocampal neurons will
increase production of TET1 mRNA and protein in response to activation with blue light, and that the same
neurons will increase production of DNMT1 mRNA and protein in response to corticosterone exposure at
physiological levels. The in vivo branch of this aim will look at the effect of acute stress on rats subjected to
maternal stress (MS) and those that are not. Following MS or control conditions, rats will be subjected to a forced
swim test (an acute stressor) and their hippocampi will be probed for DNMT1, TET1, and BDNF. We hypothesize
that the acute stressor will increase expression of TET1, but not DNMT1, based on the finding that following acute
stress, DNA methylation drops but DNMT1 expression does not change (Rodrigues, et al. 2015). We further
hypothesize that the acute stressor will change TET1 equally in MS and control rats, but that MS rats will show a
smaller increase in BDNF because of residual hypermethylation from their upbringing.
The proposed work is innovative because it capitalizes on prior work which has linked stress, DNA
hypermethylation, underexpression of BDNF, and depression. At the completion of this project, we will have made
significant steps toward understanding why early stress has a more lasting effect than stress later in life, how the
brain regulates expression of BDNF in response to acute stress, and how chronic stress disrupts the 5mC
equilibrium of the bdnf gene, placing the animal or person at risk of MDD. If successful, this work will provide
support for the therapeutic use of DNMT inhibitors as a class of antidepressants, potentially helping patients with
MDD for whom canonical antidepressants like tricyclics and SSRIs are ineffective.
1
Throughout we will refer to nucleic acid as bdnf, and protein as BDNF.
- 3. B. BACKGROUND AND SIGNIFICANCE
B.1. Introduction
Major depressive disorder (MDD) is the second most common mental disorder in the United States, affecting
around 16 million adults in 2012 alone (SAMSHA, 2013). Characterized by depressed mood, anhedonia, memory
impairment, and impaired social and occupational functioning, a recent estimate claims that MDD and comorbid
issues (treatment, lost productivity, other medical expenses, etc.) cost the U.S. economy $210.5 billion in 2010
(Greenberg, et al., 2015). The prognosis for those with MDD varies considerably. For the majority of patients,
available antidepressants relieve symptoms at least reasonably well. Patients with untreated or treatment
resistant MDD comprise the majority of successful suicides, which was the 10th most common cause of death in the
U.S. in 2013 (CDC, 2015). Aside from family history, chronic stress is the strongest known predictor of MDD (Caspi,
et al., 2003), but the mechanisms by which chronic stress predisposes us to MDD remains to be fully clarified.
B.2. Toward a Theory of MDD
Disentangling the causes of depression has been one of the most significant challenges in the neurobiology of
mental health in the past fifty years. The first antidepressants, monoamine oxidase inhibitors and tricyclics, were
discovered serendipitously (Zeller & Barsky, 1952; Shore & Brodie, 1952), and out of these findings, the amine
hypothesis, that depression is caused by decreased monoamine signalling, was born. The hypothesis led to
development, production, and prescription of selective serotonin reuptake inhibitors (SSRIs) and selective
norepinephrine reuptake inhibitors (SNRIs). SSRIs and SNRIs can relieve depressive symptoms, but only after a lag
period, long after they have increased synaptic monoamine levels. This suggests that rather than treating the cause
of depression, increasing amine signalling initiates a cascade that modulates the real causal agent of depression.
We now know that reuptake inhibitors increase neurotrophin levels (Molteni, et al., 2006). Increased
monoamine signalling increases postsynaptic cyclic AMP (cAMP) which leads to higher expression and activity of
the cAMP response element binding protein (CREB), a transcription factor which activates genes related to cell
growth (Menkes, et al., 1983; Nibuya, et al. 1996). One of CREB’s targets is brain-derived neurotrophic factor
(BDNF), a necessary promoter of neuronal survival and growth (Alderson, et al. 1990; Kalcheim & Gendreau,
1988). Treatment with SSRIs increases BDNF levels in the hippocampi of patients with MDD, suggesting that BDNF
can relieve symptoms of depression (Richardson, et al. 1994; Russo-Neustadt, et al., 2004).
Examination of the neurochemical abnormalities of untreated MDD further support the theory that BDNF
concentration has a role in the onset of depression. In rodent models of MDD such as the forced swim test, brain
BDNF levels are lower than normal, and BDNF infusion can alleviate behavioral despair symptoms. (Boersma, et al.
2013; Siuciak et al, 1997). Several studies in humans have found significantly reduced BDNF concentrations in the
serum of patients with severe MDD and those who have committed suicide (Kim, et al. 2007; Lee, et al. 2007; Grah,
et al. 2014; Kang, et al. 2015). It is therefore tempting to speculate that elevation of BDNF levels could treat MDD,
especially for patients with severe or treatment-resistant cases. Development of such treatments will require an
understanding of the factors that regulate BDNF’s expression. MDD has strong environmental determinants, so the
factors which regulate BDNF expression are also likely modulated by environmental stimuli.
B.3. MDD and Epigenetics
Epigenetics is the study of how cells regulate gene expression outside of the sequence of bases themselves, and is
one of the most intriguing interface points between DNA and the environment. Through attachment of different
functional groups (methyl, hydroxymethyl, acetyl, phosphate, etc.) to the DNA itself and to the histone proteins
around which DNA is wound, cells can quickly and stably alter gene expression following an upstream signal.
Because expression of BDNF can be significantly altered by environmental stimuli (such as a forced swim), it is
unsurprising that bdnf transcription is under epigenetic control. People with MDD have higher levels of DNA
methylation at promoter IV of the bdnf gene (Duclot and Kabbaj, 2015; Kang, et al., 2015). Hypermethylation at this
site can be reversed for a short time with physical exercise, which induces a mild, rapid-onset (i.e., acute) stress
response (Gomez-Pinilla, 2011). No work has yet addressed why an acute stress response relieves depression,
- 4. bd 5-mC
ACTIV INACTI
DNM
TE
Chronic
Acute
Figure 1. Schematic representation of
the differential effects of acute and
chronic stress.
while a more chronic stress response promotes it. We hypothesize that the balance of activating and repressing
epigenetic factors determines the depressive state of the individual.
B.4. DNA Methylation and Demethylation
DNA methylation is a generally repressive epigenetic patterning in which a DNA methyltransferase (DNMT) adds a
methyl group to the 5-carbon of a cytosine base that precedes a guanine (a CpG site) to produce 5-methylcytosine
(5mC). DNMT1 is the only DNMT known to be differentially regulated in response to stress, so we will focus on that
one.
DNA demethylation is a more involved process that is less fully understood. 5-methylcytosine cannot be
converted directly to cytosine, so a series of modifications replaces the methyl group with a carboxyl group, at
which point the base excision repair mechanism cuts out the carboxylated base and replaces it with an unmodified
one (Ito, et al. 2011; Rai, et al. 2008). The first step of this process, the conversion from 5-methylcytosine to 5-
hydroxymethylcytosine, is catalyzed by ten-eleven translocation methylcytosine dioxygenase 1 (TET1). While it is
only the first step in the process, current evidence suggests that TET1 is sufficient for BDNF promotion (Guo, et al.
2011; Kaas, et al. 2013), so in this study we will only consider TET1 when quantifying DNA demethylation.
Methylation of bdnf is linked to stressful events, therefore expression of TET1 and DNMT1 may also be
linked to stress. Work up to now has insufficiently disentangled the effects of acute and chronic stress on
expression of DNMTs and TET1. One group reported that in response to acute stress, DNA methylation levels
decrease without any change in DNMTs, suggesting that TET1 is upregulated (Rodrigues, et al. 2015). A second
group found that TET1 expression can be induced by artificial stimulation of neurons in cell culture (Kaas, et al.
2013). This implies that TET1 expression is coupled with action potentials in neurons, and that DNMT expression
is not. In a somewhat conflicting account, a 2013 study reported that for the two months immediately following
maternal separation (a rodent model of chronic stress) hippocampal BDNF levels were higher in stressed than in
unstressed mice, though the study did not look at levels of any DNMT or TET1 (Suri, et al., 2013). These studies did
not address the role of a chronic stress response via HPA axis activity. Contradicting this, prenatal stress and
corticosterone injection (which is normally elevated during chronic stress) were both found to decrease
expression of BDNF in rat hippocampi (Dong, et al. 2015; Schaaf, et al. 1998). In light of this, we hypothesize that
action potentials upregulate production of TET1 and corticosterone exposure upregulates production of DNMT1.
B.5. A Model of Stress-Induced MDD
The reactive nature of the methylation status on the bdnf gene lends itself to a
model of DNMT/TET1 antagonism based on chemical equilibrium, here called
5mC equilibrium. The position of that equilibrium is determined by relative
concentrations of DNMTs and TET1 while the actual rates of turnover
(effectively kon and koff) are determined by expression level of those genes. Our
model posits that immediately following a stressful event, activity in the
hippocampus increases. This increases expression of TET1, which in turn
elevates BDNF levels by replacing and removing methyl groups by the
mechanism described above. The equilibrium position has shifted to the left.
Following the onset of a stressor, cortisol in the blood activates glucocorticoid
receptors, increasing expression of DNMTs, which replace the methyl groups
stripped off by the TET1 pathway, returning the bdnf gene to its baseline
methylation status. This allows the organism to react adaptively to acutely stressful situations. In cases of chronic
stress, cortisol levels remain high for a considerable time, pushing the 5mC equilibrium past a healthy baseline.
Chronic stress is known to predispose people to depression, and according to this model, this is because chronic
stress produces chronically high levels of DNMTs, leading to underproduction of BDNF.
Immediately following birth, BDNF and DNMTs are highly expressed (Simmons, et al., 2013). Because of the
repressive effect of DNMTs on BDNF expression, an activating enzymatic force must also be present. We theorize
that the TET1 pathway must also be elevated in the neonatal brain; Aim 1 address this. This would produce a high
methyl turnover rate. As the brain matures, expression of DNMTs declines, as does expression of BDNF, which
- 5. suggests a parallel downregulation of the TET1 pathway. The neonatal phase can thus be seen as the period in
which the baseline methylation level is set. Should the balance of methylating and demethylating factors fall out of
balance during this critical phase, the shift in 5mC equilibrium position may be harder to change once the turnover
rate has decreased to adulthood levels.
B.6. Significance
If our hypotheses are confirmed, we will have made significant progress toward understanding the model of
depression as a dysregulation of bdnf methylation status. While this is almost certainly not a valid explanation for
every case of MDD, it opens up new possibilities for treatment which may provide relief for those who do not
respond to currently available antidepressants. Our work may also contribute to the development of diagnostic
biomarkers for MDD. Serum BDNF concentration is an unreliable metric because it also predicts schizophrenia,
bipolar disorder, and even heart failure (Oral, et al., 2012; Xiong, et al., 2014; Fukushima, et al., 2015). Serum bdnf
mRNA methylation has been shown to reliably reflect bdnf methylation status in the brain, but assaying for DNA
methylation is expensive and time-consuming (Kundakovic, et al. 2014). If we can show that relative levels of
serum DNMT1 and TET1 correlate with MDD alone, we may be closer to a laboratory diagnostic test for MDD.
C. RESEARCH DESIGN AND METHODS
C.0. Background Methods
C.0.a. Animals
The rats used in this study will be kept at Oberlin College and treated in strict accordance with guidelines set forth
by the Animal Care and Use Committee. Because our study examines the impact of environmental conditions on
genetically wild-type rats, we will use standard outbred Sprague Dawley rats for all experiments requiring whole
animals. All behavioral testing will take place in the secure behavioral testing suite.
C.0.b. Cell Culture
We will obtain primary rat hippocampus neurons from Life Technologies (Catalog #A10841-01). Plasmid DNA will
be acquired from the Karl Deisseroth lab (AddGene Plasmid 20938), which contains Channelrhodopsin-2 with a
sensitising mutation fused to mCherry for visual selection, and Ampicillin resistance and Kanamycin resistance for
chemical selection. Cells will be transfected with a mixture of all three constructs using the jetPEI transfection
system (manufactured by PolyPlus) according to the published protocol and specific information provided for rat
neurons. We will select for transfected cells with ampicillin, and verify the success of the selection by examining for
mCherry fluorescence under a light microscope.
C.0.c. Chronic Stress: Maternal Separation
Maternal separation from the dam has been shown to have a wide variety of behavioral consequences later in life,
including changes in BDNF expression in rats (Suri, et al. 2013). Here we will follow the maternal separation
procedure used by the above paper to induce early chronic stress to facilitate comparison to other studies which
did the same. We will separate male pups from the dams for 3 hours per day, from postnatal day 2 (P2) to P14. This
is significantly more separation than a pup would experience normally (Nylander & Roman, 2013) but still allows
for appropriate nutrition and physical maturation.
C.0.d. Acute Stress: Forced Swim Test
The forced swim test is a widely accepted model of acute and chronic stress and has been extensively used to test
the efficacy of new antidepressants (Porsolt, et al. 1977). If the animal receives several forced swims, signs of
behavioral despair and neurochemical changes similar to MDD appear in the animal, both of which can be
alleviated with antidepressants (Petit-Demouliere et al., 2005). Our rats will only receive one 15 minute forced
swim to model an acutely stressful event.
C.0.e. Modelling high cortisol levels
In order to model the effects of chronic stress, one of which is high circulating cortisol, without first triggering an
acute stress response, we will inject rats with corticosterone (Sigma-Aldrich) at a dosage of 1.7 mg/mL of blood
volume (Roceri, et al. 2004). Roceri’s group observed a serum cortisol concentration of 1.7 mg/mL following a
stressful event. Blood volume (BV) in mL will be calculated according to the formula BV = 0.06 * body mass (g) +
- 6. 0.77 (Lee and Blaufox, 1985). Cells in culture will receive the same dosage to mimic serum levels they would
encounter in the brain.
C.0.e. Quantifying mRNA
To help determine changes in total expression of the genes of interest, we will examine mRNA levels of dnmt1, tet1,
and bdnf and quantify their relative expression by quantitative PCR (qPCR). Because the study is concerned only
with the effects of stress on the hippocampus, in all cases we will isolate hippocampal tissue before performing
qPCR. Because our proposed experiments may alter neurite growth, we will use both Actin and Lactate
dehydrogenase A as housekeeping proteins to avoid potential bias in our control.
C.0.f. Quantifying protein
To quantify protein expression levels we will use Western blotting, which separates proteins on the basis of
molecular weight and charge. We will stain for Actin and LDH-A as housekeeping proteins and using densitometry
each experimental enzyme will be normalized to actin and then to LDH-A before comparison between time points
using a Student’s t-test.
C.1. Aim 1 - To characterize the expression profile of TET1 in the neonatal rat hippocampus
C.1.a. Hypothesis
We hypothesize that expression levels of TET1 in the neonatal rat hippocampus will follow a pattern similar to that
of DNMTs in the neonatal rat hippocampus as reported by Simmons et al. 2013.
C.1.b. Rationale
Recent work by Simmons et al. (2013) showed that DNMT1, DNMT3a, and DNMT3b are highly expressed in
developing rat brains immediately following birth and that these expression levels decrease significantly within 3
months. The group also found that accompanying this drop in expression of DNMTs, global methylation levels in
the hippocampus increase. The fact that 5mC levels gradually increase even as methylator expression decreases
suggests that demethylating enzymes like TET1 have high expression levels initially and tail off in a similar but
exaggerated fashion to DNMTs as the rat develops adulthood. No research has yet examined the expression profile
of TET1 and BDNF in neonatal rats. If we can confirm that TET1 levels do follow this trajectory, this lends support
to the general hypothesis that the neonatal hippocampus undergoes rapid turnover of DNA methylation which
gradually slows as the animal develops.
C.1.c. Experimental design
To facilitate comparison between studies we will use the same time points (P1, P4, P7, P10, P14, P21, P75) used by
Simmons, et al. in their 2013 investigation of DNMT expression levels. The previous study looked at DNMT1,
DNMT3a, and DNMT3b in a variety of brain regions. We will only look at hippocampal tissue, and will probe for
BDNF, DNMT1 (to replicate Simmons, et al’s results) and TET1. Rats will be sacrificed and hippocampal tissue
extracted and sonicated. We will probe for tet1, dnmt1, and bdnf mRNA levels by qPCR and protein levels with
Western blotting.
C.1.d. Interpretation of Results and Alternative Results
We expect to find that TET1 has an expression profile similar to DNMT1, in which expression immediately
following birth is quite high but tails off to lower, stable levels by P21 (Simmons, et al, 2013). Because TET1 and
DNMT1 have opposite effects in regulating BDNF expression, confirmation of this hypothesis will provide evidence
for rapid DNA methylation turnover in the neonatal brain and for why changes in 5mC equilibrium that arise
during early life persist into adulthood. If expression levels are much lower in adulthood, a larger stimulus would
be necessary to shift 5mC equilibrium. We will look for a significant difference in protein expression between P1
and P21 and between P1 and P75 using Student’s t-test. There are a number of alternative outcomes of this aim.
We may find that TET1 levels remain relatively low; this would argue for a third actor in the system which
“protects” 5hmC on the genome. The 5mC binding protein MeCP2 could fill this role; it has been shown to bind both
5mC and 5hmC, and to help maintain the activity status of either modification (Mellén, et al. 2012).
C.1.e. Potential Pitfalls and Alternative Approaches
- 7. This aim uses the approach of a previous paper in order to fill in a gap in knowledge which will contribute to our
proposed model. Regardless of the outcome, uncovering the expression profile of TET1 will be useful in a number
of different branches of developmental neuroscience.
C.2. Aim 2: To examine the effect of acute and chronic stress on BDNF, TET1, and DNMT1 expression
C.2.a. Hypothesis
We hypothesize that TET1 production is linked to neural activation, producing a fast response to stress and that
DNMT1 production is linked to the slower endocrine stress response. We propose to test this hypothesis using in
vitro and in vivo models.
C.2.b. Rationale
(see paragraph 3 of B.4.)
C.2.c. Experimental Design
C.2.c.i. In vitro
We will culture rat hippocampal neurons expressing channelrhodopsin-2. Neurons will be exposed to pulses of
blue light. At 0, 5, 10, 30, 60, 120, and 360 minutes following exposure, cells will be concentrated, lysed, and
probed for TET1, DNMT1, and BDNF mRNA and protein (by qPCR and Western blotting respectively). An identical
set of cells will receive corticosterone at a dosage of 1.7 mg/mL into the solution. At the same timepoints, these
cells will be concentrated, lysed, and probed.
C.2.c.ii. In vivo
We will use eight groups of 10 rats each for this study. Four groups will have no stress, and half will be subjected to
Maternal Separation (MS) as described in C.0.c. At day 75, one group from each stress condition will be assigned a
treatment: no treatment, a saline injection, a corticosterone injection to bring blood volume to 1.7 mg/mL, or the
forced swim test for 15 minutes. Two hours after the treatment, rats will be sacrificed, the hippocampus will be
extracted and homogenized, and we will probe for TET1, DMNT1, and BDNF mRNA and protein by qPCR and
Western blotting.
C.2.d. Interpretation of Results and Alternative Results
We expect the optogenetically stimulated neurons to show a rapid spike in TET1 expression and the corticosterone
exposed neurons to show a slower upregulation of DNMT1. We further expect that each group of MS rats will have
lower BDNF expression than their unstressed cohorts, but that in response to acute stress and cortisol, expression
levels of DNMT1 and TET1 will follow roughly the same course as the unstressed mice. We hope to show that TET1
levels increase significantly from their baseline, then return to their baseline relatively quickly, using Student’s t-
test. We also hope to show a slower but significant upregulation of DNMT1, validated by the same means. We may
see an effect in which MS increases expression of both TET1 and DNMT1, which would fall in line with Dong et al.
2015’s paper which used prenatal stress as a model. This finding would suggest that MS increases baseline
expression of TET1 and DNMT1, rendering BDNF levels more sensitive to changes in either’s expression. Because
cortisol persists longer than high neural activity, this could explain the tendency for MS mice to have low BDNF
levels later in life.
C.2.e. Potential Pitfalls and Alternative Approaches
Mammalian cell culture is temperamental and sometimes difficult to maintain. Keeping consistency from batch to
batch of culture will be vital to the success of the experiment. If the results are promising, we hope to monitor the
enzymes’ location using fluorescently tagged TET1 and DNMT1. Most significantly, we acknowledge the limited
scope of the hypothesized model. Using a simple hypothetical system allows us to more easily rule out components
and reformulate it if necessary. Future work will combine the findings of our proposed study with outside data to
produce a more complete picture of the link between BDNF and stress.
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